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In order to study the involvement of chitinases in the interactions between grape berries and fungi, three genomic sequences encoding chitinases were isolated by PCR walking on Vitis vinifera DNA. Two of these belong to class I chitinases with a putative vacuolar (Vvchit1a) and extracellular (Vvchit1b) localization whilst the third sequence belongs to class III (VvchitIII). Transcripts of class III chitinases were shown to accumulate in unripe berries infected with Plasmopara viticola but not at later developmental stages of berries infected with Uncinula necator or Botrytis cinerea. In contrast, class I transcripts were never detected in grape berries. Specificity of chitinase expression in response to pathogens was investigated in leaves infected with B. cinerea or Pseudomonas syringae pv. pisi, a bacterium leading to an incompatible interaction. Expression of the Vvchit1a gene was induced in leaves upon both infections. In contrast, accumulation of class III transcripts was observed in response to P. syringae but not to B. cinerea infection, whereas Vvchit1b transcripts were detected in leaves only in the latter case. Our data indicate that, in V. vinifera, induction of chitinase genes depends on the infecting pathogen.

In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which approximately 50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors.

In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions., Author Summary Despite the essential functions of roots in plant access to water and nutrients, root system architecture has not been directly considered for crop breeding improvement, but it is now considered key for a “second green revolution.” In this study, we aimed to decipher integrated molecular mechanisms coordinating lateral organ development in legume roots: lateral roots and nitrogen-fixing symbiotic nodules. The compact root architecture 2 (cra2) mutant form an increased number of lateral roots and a reduced number of symbiotic nitrogen-fixing nodules. This mutant is affected in a CLAVATA1-like Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that has not previously been linked to root development. Grafting experiments showed that CRA2 negatively controls lateral root formation and positively controls nodule development through local and systemic pathways, respectively. Overall, our results can be integrated in the framework of regulatory pathways controlling the symbiotic nodule number, the so-called “Autoregulation of Nodulation” (AON), involving another LRR-RLK that also acts systemically from the shoots, SUNN (Super Numeric Nodules). A coordinated function of the CRA2 and SUNN LRR-RLKs may thereby permit the dynamic fine tuning of the nodule number depending on the environmental conditions.

To investigate how exudation shapes root-associated bacterial populations, transgenic Arabidopsis thaliana plants that exuded the xenotopic compound octopine at low and high rates were grown in a nonsterile soil. Enumerations of both cultivable and octopine-degrading bacteria demonstrated that the ratios of octopine degraders increased along with octopine concentration. An artificial exudation system was also set up in which octopine was brought at four ratios. The density of octopine-degrading bacteria directly correlated with the input of octopine. Bacterial diversity was analysed by rrs amplicon pyrosequencing. Ensifer and Pseudomonas were significantly more frequently detected in soil amended with artificial exudates. However, the density of Pseudomonas increased as a response to carbon supplementation while that of Ensifer only correlated with octopine concentrations possibly in relation to two opposed colonization strategies of rhizosphere bacteria, that is, copiotrophy and oligotrophy.

International audience; Rhizobia and legumes establish symbiotic interactions leading to the production of root nodules, in which bacteria fix atmospheric nitrogen for the plant's benefit. This symbiosis is efficient because of the high rhizobia population within nodules. Here, we investigated how legumes accommodate such bacterial colonization. We used a reverse genetic approach to identify a Medicago truncatula gene, SymCRK, which encodes a cysteine-rich receptor-like kinase that is required for rhizobia maintenance within the plant cells, and performed detailed phenotypic analyses of the corresponding mutant. The Medicago truncatula symCRK mutant developed nonfunctional and necrotic nodules. A nonarginine asparate (nonRD) motif, typical of receptors involved in innate immunity, is present in the SymCRK kinase domain. Similar to the dnf2 mutant, bacteroid differentiation defect, defense-like reactions and early senescence were observed in the symCRK nodules. However, the dnf2 and symCRK nodules differ by their degree of colonization, which is higher in symCRK. Furthermore, in contrast to dnf2, symCRK is not a conditional mutant. These results suggest that in M. truncatula at least two genes are involved in the symbiotic control of immunity. Furthermore, phenotype differences between the two mutants suggest that two distinct molecular mechanisms control suppression of plant immunity during nodulation.

International audience; During endosymbiotic interactions between legume plants and nitrogen-fixing rhizobia, successful root infection by bacteria and nodule organogenesis requires the perception and transduction of bacterial lipo-chitooligosaccharidic signal called Nod factor (NF). NF perception in legume roots leads to the activation of an early signaling pathway and of a set of symbiotic genes which is controlled by specific early transcription factors (TFs) including CYCLOPS/IPD3, NSP1, NSP2, ERN1 and NIN. In this study, we bring convincing evidence that the Medicago truncatula CCAAT-box-binding NF-YA1 TF, previously associated with later stages of rhizobial infection and nodule meristem formation is, together with its closest homolog NF-YA2, also an essential positive regulator of the NF-signaling pathway. Here we show that NF-YA1 and NF-YA2 are both expressed in epidermal cells responding to NFs and their knock-down by reverse genetic approaches severely affects the NF-induced expression of symbiotic genes and rhizobial infection. Further over-expression, transactivation and ChIP-PCR approaches indicate that NF-YA1 and NF-YA2 function, at least in part, via the direct activation of ERN1. We thus propose a model in which NF-YA1 and NF-YA2 appear as early symbiotic regulators acting downstream of DMI3 and NIN and possibly within the same regulatory complexes as NSP1/2 to directly activate the expression of ERN1.

Background: Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking. Results: In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 miRtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions. Conclusions: We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops. Fil: Formey, Damien. Centre National de la Recherche Scientifique; Francia. Université de Toulouse; Francia Fil: Sallet, Erika. Institut National de la Recherche Agronomique; Francia Fil: Lelandais Brière, Christine. Université Paris Diderot - Paris 7; Francia Fil: Ben, Cécile. Université de Toulouse; Francia Fil: Bustos Sanmamed, Maria del Pilar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Université Paris Diderot - Paris 7; Francia Fil: Niebel, Andreas. Institut National de la Recherche Agronomique; Francia Fil: Frugier, Florian. Centre National de la Recherche Scientifique; Francia Fil: Combier, Jean Philippe. Centre National de la Recherche Scientifique; Francia Fil: Debellé, Frédéric. Institut National de la Recherche Agronomique; Francia Fil: Hartmann, Caroline. Université Paris Diderot - Paris 7; Francia Fil: Poulain, Julie. Commissariat A Energie Atomique; Francia Fil: Gavory, Frédérick. Commissariat A Energie Atomique; Francia Fil: Wincker, Patrick. Commissariat A Energie Atomique; Francia Fil: Roux, Christophe. Centre National de la Recherche Scientifique; Francia Fil: Gentzbittel, Laurent. Université de Toulouse; Francia Fil: Gouzy, Jérôme. Institut National de la Recherche Agronomique; Francia Fil: Crespi, Martin. Université Paris Diderot - Paris 7; Francia

International audience; Downy mildew is a major disease of grapevine caused by the oomycete Plasmopara viticola. It is at the origin of numerous fungicide treatments. In order to optimize these treatments and reduce their number, an early detection of the disease in the field is sought-after, preferably by non-destructive means for a precision agriculture approach. Stilbenes, the main phytoalexin of grapevine, are not present in healthy leaves. Their synthesis and accumulation is induced by P. viticola. They are fluorescent phenolic compounds, displaying violet-blue fluorescence (VBF). In the laboratory, VBF was used to assess stilbenes in vivo in grapevine leaves or as an indicator of the development of the infection by P. viticola. So, this fluorescence signal could potentially be used as a non-invasive proxy for the presence of downy mildew. A new portable sensor for stilbene VBF, Mx-330, was developed by the company FORCE-A (Orsay, France). The present study confirmed that this new sensor could be used in the vineyards to detect downy mildew. Thanks to its presence also on the adaxial leaf side, P. viticola-induced stilbene VBF is suitable for vehicle-mounted proximal sensing.

International audience; Two austenitic stainless steels, AISI 304L and AISI 303, containing 0.025 and 0.249 wt%S were oxidized in thermobalance at 1000°C for 50h. The chemical composition and the crystallographic structure of the oxide scales were investigated by Raman spectroscopy. Adhesion of oxide scales was tested by SEM in situ tensile tests. A correlation between the specific mass change, the chemistry, the microstructure and the adhesion properties is made and results are discussed in relation with sulphur concentration in the alloy.

In this multidisciplinary study, we combined morphological, physiological, and phylogenetic approaches to identify three dominant water bloom-forming Cyanobacteria in a tropical marine mangrove in Guadeloupe (French West Indies). Phylogenetic analysis based on 16S rRNA gene sequences place these marine Cyanobacteria in the genera Oscillatoria ( Oscillatoria sp. clone gwada, strain OG ) or Planktothricoides (‘ Candidatus Planktothricoides niger’ strain OB and ‘ Candidatus Planktothricoides rosea’ strain OP ; both provisionally novel species within the genus Planktothricoides). Bioassays showed that ‘ Candidatus Planktothricoides niger’ and ‘ Candidatus Planktothricoides rosea’ are toxin-producing organisms. This is the first report of the characterization of Cyanobacteria colonizing periphyton mats of a tropical marine mangrove. We describe two novel benthic marine species and provide new insight into Oscillatoriaceae and their potential role in marine sulfide-rich environments such as mangroves.

International audience; Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites. Four nucleus-encoded VDACs have been identified in Arabidopsis thaliana. Alternative cleavage and polyadenylation generate two VDAC3 mRNA isoforms differing by their 3' UTR. By using quantitative RT-PCR and in vivo mRNA visualization approaches, the two mRNA variants were shown differentially associated with mitochondria. The longest mRNA presents a 3' extension named alternative UTR (aUTR) that is necessary and sufficient to target VDAC3 mRNA to the mitochondrial surface. Moreover, aUTR is sufficient for the mitochondrial targeting of a reporter transcript, and can be used as a tool to target an unrelated mRNA to the mitochondrial surface. Finally, VDAC3-aUTR mRNA variant impacts mitochondria morphology and size, demonstrating the role of mRNA targeting in mitochondria biogenesis.

Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H+-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H+-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H+-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H+-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells.

International audience; Rhizobia and legumes are able to interact in a symbiotic way leading to the development of root nodules. Within nodules, rhizobia fix nitrogen for the benefit of the plant. These interactions are efficient because spectacularly high densities of nitrogen fixing rhizobia are maintained in the plant cells. DNF2, a Medicago truncatula gene has been described as required for nitrogen fixation, bacteroid's persistence and to prevent defense-like reactions in the nodules. This manuscript shows that a Rhizobium mutant unable to differentiate is not sufficient to trigger defense-like reactions in this organ. Furthermore, we show that the requirement of DNF2 for effective symbiosis can be overcome by permissive growth conditions. The dnf2 knockout mutants grown in vitro on agarose or Phytagel as gelling agents are able to produce nodules fixing nitrogen with the same efficiency as the wild-type. However, when agarose medium is supplemented with the plant defense elicitor ulvan, the dnf2 mutant recovers the fix- phenotype. Together, our data show that plant growth conditions impact the gene requirement for symbiotic nitrogen fixation and suggest that they influence the symbiotic suppression of defense reactions in nodules.

Since the early 1990s, various strategies have been proposed to rehabilitate trace element (TE) polluted areas by phytoremediation. Among these strategies, phytoextraction consists in TE uptake from soil and accumulation by plants. To implement this strategy, it has been proposed to use poplar due to its fast growth, its large biomass and its use in energy production. However, a substantial proportion of absorbed metals is accumulated in poplar leaves which fall in autumn. Thus, poplar phytoextraction efficiency may be limited if TE are not re-mobilized during autumn senescence. In this context, part of this thesis work has been carried out on the experimental field of Pierrelaye which was polluted by the spreading of sewage water from Paris. Among the 14 poplar genotypes growing on the field, we tried to identify those that efficiently remobilize leaf metals to perennial organs. Metal content, gene expression and correlative analyses have been undertaken, providing new insight in the management of metals in leaves. Because the vacuole is the main metal storage compartment in the cell, NRAMP (Natural Resistance-Associated Macrophage Protein) vacuolar efflux proteins previously identified in Arabidopsis thaliana are good candidates to enhance leaf metal remobilization. Characterization of their homologues in poplar was therefore undertaken by expression in yeast and in A. thaliana. In order to independently control the transport of essential and non-essential metals by NRAMP, a study aiming to identify the structural determinants involved in selectivity was undertaken. Expression of NRAMP mutants affected in their selectivity in A. thaliana highlighted their impact on metal accumulation and tolerance. To study the involvement of more general nutrient recycling mechanisms in metal remobilization during leaf senescence, the involvement of autophagy was tested in A. thaliana. Physiological characterization of autophagy deficient plants indicated that this mechanism plays a role in metal use efficiency and probably in metal remobilization during senescence. By combining a field approach on poplar and molecular genetics in Arabidopsis, this work opens multiple perspectives to specifically reduce the accumulation of TE in poplar leaves., Depuis le début des années 1990, différentes stratégies de phytoremédiation ont été proposées pour réhabiliter les zones polluées par des éléments traces métalliques (ETM). Parmi ces stratégies, la phytoextraction consiste en l’absorption et l’accumulation par les plantes des ETM présents dans les sols. Afin de mettre en place cette stratégie, il a été proposé d’utiliser le peuplier en raison de sa croissance rapide, de son importante biomasse et de ses débouchés énergétiques. Cependant une proportion considérable des métaux absorbés par cet arbre est accumulée dans les feuilles alors que celles-ci chutent à l’automne. Ainsi, l’efficacité de phytoextraction du peuplier peut se trouver limitée si aucun mécanisme de remobilisation des ETM n’est mis en place au cours de la sénescence automnale. Dans ce contexte, une partie des travaux de cette thèse a été réalisée sur la parcelle expérimentale de Pierrelaye polluée suite à l’épandage d’eaux usées de la ville de Paris. Nous avons recherché parmi les 14 génotypes de peuplier présents sur le site, ceux qui sont les plus efficaces pour remobiliser les métaux des feuilles vers les parties pérennes. Des mesures de contenu en métaux, d’expression de gènes et des analyses corrélatives ont ouvert de nouvelles pistes concernant la gestion des métaux foliaires. Parce que la vacuole constitue le principal lieu de stockage des métaux de la cellule, les protéines d’efflux vacuolaire NRAMP (Natural Resistance-Associated Macrophage Protein) précédemment identifiées chez Arabidopsis thaliana, représentent de bons candidats pour stimuler la remobilisation des métaux foliaires. La caractérisation de leurs homologues chez le peuplier a donc été entreprise par expression chez la levure et chez A. thaliana. Afin de contrôler indépendamment le transport des métaux essentiels et non-essentiels chez les NRAMP, une étude visant à identifier les déterminants structuraux impliqués dans leur sélectivité a été réalisée. La caractérisation des mutants NRAMP affectés dans leur sélectivité par expression chez A. thaliana a mis en lumière leur impact sur l’accumulation et la tolérance aux métaux. Dans le but d’étudier l’implication de mécanismes plus généraux de recyclage des nutriments dans la remobilisation des métaux au cours de la sénescence foliaire, le rôle de l’autophagie a été testé chez A. thaliana. L’étude de plantes déficientes pour l’autophagie a montré l’implication de ce mécanisme dans l’efficacité d’utilisation des métaux et probablement dans leur remobilisation au cours de la sénescence. En combinant des études en champ sur le peuplier et de génétique moléculaire chez Arabidopsis, ce travail permet de proposer différentes pistes pour diminuer spécifiquement l’accumulation des ETM dans les feuilles de peuplier.

International audience; PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen-fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M.truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M.truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down-regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down-regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants.

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

Genome size and base composition in 16 species and subspecies of the Hydrangea, a woody ornamental genus of Hydrangeaceae, were evaluated by flow cytometry in relation to their chromosome number. This is the first such study concerning the genome size of these species together with a karyotype study of the most important species, Hydrangea macrophylla subsp. macrophylla (Hortensia), from an economical point of view. The 2C DNA content ranged from 1.95 pg in Hydrangea quercifolia to 5.00 pg in Hydrangea involucrata. The base composition ranged from 39.9% GC in Hydrangea aspera subsp. sargentiana to 41.1% in Hydrangea scandens subsp. scandens (significant difference at p < 0.05). The smallest genome sizes were those of the three species originating from North or South America. Most of the species studied presented a chromosome number of 2n = 2x = 36, except for those of the section Aspereae which showed 2n = 30, 34 and 36. A primary karyotype has been made for the first time for H. macrophylla subsp. macrophylla. Phylogenetic relationships between species, the origin of chromosome number and an exploration of the genetic diversity within the genus are discussed.

Recent work has assessed the potential of peptide deformylase (PDF) as a target for broad spectrum antibacterial agents. By using a number of approaches, including proteomics, researchers at Roche have shown that the molecules they had selected in vitro were able to target PDF in vivo. However, the authors, having observed resistance occurring at a rather high frequency and on the basis of the recent discovery of a deformylase homologue in humans, suggest that PDF 'may not be an optimal target for broad spectrum antibacterial agents'. We link these data to results published by other laboratories and conclude that PDF deserves to still be considered a valuable target for new antibiotics.

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513–515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.

To avoid polyploidy in regenerants the source of explant material should be monosomatic. Therefore, the leaf and petiole tissue of five diploid Medicago species (Medicago ciliaris, Medicago murex, Medicago orbicularis, Medicago polymorpha and Medicago truncatula cv. Jemalong, and the ecotype R108-1) was assessed for polysomaty by flow cytometry. For the species studied the frequency of 2C nuclei was about 90% in leaves compared with that in petioles. Embryos were readily formed from tissue of leaves in liquid media containing 1 mg l(-1) or 4 mg l(-1) dichlorophenoxyacetic acid (2,4-D). For embryo development two procedures were tested - prolonged use of induction medium and treatment with polyethylene glycol Mw 6000 (PEG). The highly regenerable genotypes M. truncatula cv. Jemalong and R108-1 showed efficient conversion of embryos after maturation in liquid medium. The regenerated plants were diploid and with normal phenotype.

We have previously shown that in Nicotiana sylvestris cytoplasmic male-sterile (CMS) mutants where the mtDNA lacks the nad7 gene coding for a subunit of respiratory Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), glycine (Gly) oxidation was lower than in the wild type and insensitive to rotenone, suggesting Complex I dysfunction. In contrast, the oxidation rate of exogenous NADH and the capacity of the cyanide-resistant respiration (AOX) were enhanced. Here we report that, in contrast to Gly, the rate of malate oxidation was not affected, but proceeded totally in a rotenone-insensitive pathway, strongly suggesting that survival of CMS plants depends on the activation of internal and external alternative NAD(P) H dehydrogenases and that Gly decarboxylase activity depends on Complex I functioning. A similar defect in Complex I activity and Gly oxidation was found in the NMS1 nuclear mutant, defective in the processing of the nad4transcript, but alternative NAD(P) H dehydrogenases were less activated. In CMS and NMS1, the fraction of the AOX pathway was increased, as compared to wild type, associated with higher amounts ofaox transcripts, AOX protein, and plant resistance to cyanide. Non-phosphorylating respiratory enzymes maintained normal in vivo respiration levels in both mutants, but photosynthesis was decreased, in correlation with lower leaf conductance, emphasizing mitochondrial control on photosynthesis.

Faba bean necrotic yellows virus (FBYNV), Milk vetch dwarf virus (MDV), and Subterranean clover stunt virus (SCSV) are nanoviruses that infect leguminous plants. From MDV- and SCSV-infected tissue we identified viral DNAs that encode a replication initiator protein (Rep), essential for replication of the multiple circular single-stranded DNAs of these viruses. These previously undescribed Rep proteins of MDV and SCSV are strikingly similar in sequence and functionally equivalent to the master Rep protein of FBYNV. Moreover, we demonstrated that the master Rep proteins of the three viruses are able to trigger replication of heterologous nanovirus DNAs. Such cross-species replication may reflect a considerable potential for genetic reassortment among nanoviruses in nature and be of significance for their evolution.

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.

Two protein kinases displaying mitogen-activated protein kinase (MAPK) properties are activated both by an hypoosmotic stress and by oligogalacturonides in tobacco cell suspensions [Cazalé et al. (1999) Plant J. 19, 297–307]. Using specific antibodies, they were identified as the salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK). The SIPK was also activated by an hyperosmotic stress, indicating that the same kinase may play a role both in hypo- and hyperosmotic signalling pathways, in addition to its involvement in the transduction of elicitor signals. Using immunoprecipitation followed by two-dimensional in-gel kinase assay, three molecular forms of the SIPK were observed, suggesting that additional modifications of the activated kinase may occur. In contrast to WIPK and SIPK, which are located at the crossroad of several transduction pathways initiated by elicitor or osmotic stimuli, a 44 kDa kinase, that would not belong to the MAPK family, appeared more specific to osmotic stress.

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.

Nod factors are important elicitors in legume-bacterium symbiosis. Any candidate plant receptor(s) for these lipo-oligosaccharides can be expected to show some lectin-like properties. A novel protein (P60), a native tetramer with 60 kDa monomers, has been isolated from a membrane fraction of Medicago sativa (lucerne, alfalfa) roots by using affinity chromatography with either GlcNAc or N,Nʹ,Nʹʹ-triacetyl-(1 → 4)-β-D-chitotriose [(GlcNAc)3] grafted to agarose beads as the matrix and, in a second step, Sephadex G-200 gel filtration. With (GlcNAc)3-agarose an additional protein of 78 kDa was isolated. P60 showed haemagglutination activity with specificity for GalNAc, GalN, GlcNAc and GlcN. Binding experiments with radioactive GlcNAc gave a Kd of 95 nM and one binding site per monomer of P60; Nod factor competed strongly for this binding. In native PAGE, protein incubated with O-sulphated Nod factors had a higher electrophoretic mobility as a consequence of binding. However, the largest modification was observed with a natural mixture of Nod factors, containing the O-acetylated and O-sulphated tetrasaccharidic NodRm-IV(Ac,S) (in which Ac stands for an O-acetylated group at the non-reducing end and S for O-sulphation at the reducing end) in addition to the non-O-acetylated NodRm-IV(S) (which alone had little effect) and NodRm-V(S). The native PAGE study was also performed with known lectins from other sources, but only the 34 kDa lectin of Phytolacca americana (pokeweed) showed any such interaction, although without discrimination between Nod factors. Finally, one peptide of each isolated protein was sequenced; the peptide from P60 showed some similarity with dihydrolipoamide dehydrogenase and ferric leghaemoglobin reductase, whereas the peptide from P78 was identical with an analogous region of 70 kDa heat shock protein.

The progression of cell cycle in eukaryotes is controlled by protein complexes composed of p34 protein kinase and cyclin subunits. Recently, we have described four B1 type mitotic cyclin in yellow lupine. The presence of several closely related cyclin genes within the same plant species raised the question about tissue specificity of respective cyclins or their different regulations by plant-specific signals. Therefore, we examined the expression pattern of four B1 cyclins in various lupine tissues, with special emphasis put on developing root nodules. We also studied the effect of phytohormones on the level of respective cyclin mRNAs. As expected, cyclin transcript accumulation was restricted to proliferating tissues. Detailed analysis by reverse transcription-PCR and using primers specific to each cyclin allow to establish that different genes are engaged in cell divisions of various meristematic tissues. All four genes were activated during nodule development, however, the Cyc3 and Cyc4 genes mostly at the early stages of nodulation, whereas the Cyc1 and Cyc2 genes within the mature nodule organs. The expression of cyclins was regulated differently by plant growth factors. Both auxin and cytokinin induced the Cyc1 and Cyc4 genes and their transcript level was also abundant within the root, shoot and floral meristems. The precise localisation of cyclin transcripts by in situ hybridisation revealed that lupine nodule meristem was active during the whole process of nodule development, even in old organs. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Summary Hypoosmotic stress activates a phosphorylation-dependent oxidative burst. In-gel kinase assays were performed to characterize the protein kinases that could be implicated in osmoregulation and in the activation of the oxidative burst. Hypoosmotic stress activated several kinases among which 50 and 46 kDa proteins displayed mitogen-activated protein kinase (MAP kinase) properties. They phosphorylated myelin basic protein in the absence of calcium, were recognized by antibodies directed against human MAP kinases, and were phosphorylated on tyrosine. Immunoprecipitation with an antibody directed against the tobacco MAP kinase Ntf4 showed that at least one of the activated kinases would be Ntf4-like. Apigenin, a MAP kinase and cyclin-dependent kinase inhibitor which prevents the hypoosmotically induced oxidative burst ( Cazaleet al. 1998 ; Plant Physiol. 116, 659–669), inhibited these kinases in vitro suggesting that they may play a role in the activation of the oxidative burst. Like the oxidative response, activation of the kinases depended on extracellular calcium influx and protein kinases sensitive to staurosporine and 6-DMAP. However, kinase activation did not depend on effluxes through anion channels or on the oxidative burst. Two-dimensional in-gel kinase assays revealed the presence of three protein kinases with an apparent molecular mass of 50 kDa and one of 46 kDa, all four being activated by hypoosmotic stress. The same kinases were also activated by oligogalacturonides and salicylic acid, underlying the importance of these MAP kinases as common components of different signaling pathways triggered by different extracellular stimuli.

The genome size and the base composition (GC%) of eight Hypochaeris species were determined by flow cytometry in order to establish the pattern of nuclear DNA variation within the genus. The species analysed showed an almost fivefold range of variation from 1.68 pg in H. cretensis to 8.10 pg in H. uniflora. This variation in DNA content is greater between taxonomic sections of Hypochaeris species than within a section. There was no correlation between 2C DNA content and GC% indicating that neither the GC fraction nor the AT fraction were preferentially associated with variation in genome size. Because there is little heterochromatin, these results show that it is interspersed repeated sequences that are most probably implicated in this variation. From phylogenetic analysis, it is likely that genome size has evolved by loss of DNA content in some lineages and by gain in one lineage from an ancestral genome which was probably similar to genomes of intermediate size in Hypochaeris.

International audience; In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/Luxl class. The Luxl-like protein Tral synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/Lux1 type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids.

International audience; [FeFe]-hydrogenase-like genes encode [Fe4S4]-containing proteins that are ubiquitous in eukaryotic cells. In humans, iron-only hydrogenase-like protein 1 (IOP1) represses hypoxia inducible factor-1 subunit (HIF1-) at normal atmospheric partial O-2 pressure (normoxia, 21kPa O-2). In yeasts, the nar1 mutant cannot grow at 21kPa O-2,O- but can develop at a lower O-2 pressure (2kPa O-2). We show here that plant [FeFe]-hydrogenase-like GOLLUM genes are essential for plant development and cell cycle progression. The mutant phenotypes of these plants are seen in normoxic conditions, but not under conditions of mild hypoxia (5kPa O-2). Transcriptomic and metabolomic experiments showed that the mutation enhances the expression of some hypoxia-induced genes under normal atmospheric O-2 conditions and changes the cellular content of metabolites related to energy metabolism. In conclusion, [FeFe]-hydrogenase-like proteins play a central role in eukaryotes including the adaptation of plants to the ambient O-2 partial pressure.

Background Legumes form root nodules to house nitrogen fixing bacteria of the rhizobium family. The rhizobia are located intracellularly in the symbiotic nodule cells. In the legume Medicago truncatula these cells produce high amounts of Nodule-specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes. Results Here we used the Medicago truncatula Gene Expression Atlas (MtGEA) and other published microarray data to analyze the expression of 334 NCR genes in 267 different experimental conditions. We find that all but five of these genes are expressed in nodules but in no other plant organ or in response to any other biotic interaction or abiotic stress tested. During symbiosis, none of the genes are induced by Nod factors. The NCR genes are activated in successive waves during nodule organogenesis, correlated with bacterial infection of the nodule cells and with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. However, NCR expression is not associated with nodule senescence. According to their Shannon entropy, a measure expressing tissue specificity of gene expression, the NCR genes are among the most specifically expressed genes in M. truncatula. Moreover, when activated in nodules, their expression level is among the highest of all genes. Conclusions Together, these data show that the NCR gene expression is subject to an extreme tight regulation and is only activated during nodule organogenesis in the polyploid symbiotic cells. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-712) contains supplementary material, which is available to authorized users.

International audience; Alternative splicing (AS) of pre-mRNA represents a major mechanism underlying increased transcriptome and proteome complexity. Here, we show that the nuclear speckle RNA-binding protein (NSR) and the AS competitor long noncoding RNA (or ASCO-IncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change in AS patterns of many genes, a response largely dependent on NSRs. RNA immunoprecipitation assays demonstrate that AtNSRs interact not only with their alternatively spliced mRNA targets but also with the ASCO-RNA in vivo. The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. Expression of ASCO-RNA in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, IncRNA can hijack nuclear AS regulators to modulate AS patterns during development.

International audience; Fleshy fruit species such as tomato are important because of their nutritional and economic value. Several stages of fruit development such as ovary formation, fruit set, and fruit maturation have already been the subject of many developmental studies. However, fruit growth per se has been much less addressed. Fruit growth like all plant organs depends upon the developmental processes of cell division and cell expansion. The activity of cell divisions sets the number of cells that will compose the fruit; the cell expansion activity then determines its final size. Among the various mechanisms that may influence the determination of cell size, endopolyploidy by the means of endoreduplication, i.e. genome amplification in the absence of mitosis, appears to be of great importance in fleshy fruits. In tomato fruit, endoreduplication is associated with DNA-dependent cell expansion: cell size can reach spectacular levels such as hundreds of times its initial size (e.g. >0.5 mm in diameter), with as much as a 256-fold increase in nuclear DNA content. Using tomato fruit development as a model, recent investigations combining the use of flow cytometry, cellular imaging and molecular analyses have provided new data in favor of the long-standing karyoplasmic ratio theory, stating that cells tend to adjust their cytoplasmic volume to the nuclear DNA content. By establishing a highly structured cellular system where multiple physiological functions are integrated, endoreduplication acts as a morphogenetic factor supporting cell growth during tomato fruit development. In the context of plant breeding, deciphering the mechanisms controlling fruit growth, in particular those connecting the process of nuclear endoreduplication with modulation of gene expression, the regulation of cell size and final fruit size and composition, is necessary to understand better the establishment of fleshy fruit quality traits.

International audience; Plant and animals have evolved different strategies for their development. Whether this is linked to major differences in their cell mechanics remains unclear, mainly because measurements on plant and animal cells relied on independent experiments and setups, thus hindering any direct comparison. In this study we used the same micro-rheometer to compare animal and plant single cell rheology. We found that wall-less plant cells exhibit the same weak power law rheology as animal cells, with comparable values of elastic and loss moduli. Remarkably, microtubules primarily contributed to the rheological behavior of wall-less plant cells whereas rheology of animal cells was mainly dependent on the actin network. Thus, plant and animal cells evolved different molecular strategies to reach a comparable cytoplasmic mechanical core, suggesting that evolutionary convergence could include the internal biophysical properties of cells.

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens., Author Summary An ecological niche is defined, in a given environment, by the availability of nutritive resources, which can be specifically assimilated by certain living organisms to promote their proliferation. The bacterial pathogen Agrobacterium tumefaciens is able to engineer an ecological niche in the infected host via the transformation of the plant genome and diversion of the plant metabolism towards production of the opine nutrients. In this work, we quantified the selective advantage conferred to a member of the phytopathogenic species A. tumefaciens which is able to assimilate the opine nopaline. This opine is a condensate of arginine and α-ketoglurate that is produced both under linear and cyclic forms in the plant tumour environment. We further determined at the molecular and atomistic levels how A. tumefaciens is able to sense the nopaline molecules, and which metabolic pathways are activated in response. Overall, this work deciphered some key molecular events in the niche construction of the pathogen A. tumefaciens that is unique among living organisms and used to develop bioengineering tools.

Little is known about the consequences of releasing genetically engineered plants (GEP) into the environment. Using opine-producing GEP, we show that transgenic plants alter their biological environment, more precisely the root-associated bacterial populations. The alterations were both transgene-specific and target population-specific. Therefore, assessment studies on the introduction of a given transgene into a GEP will be valid on the given transgene. Evidence of any transgene-associated biological effect will depend on the determination of the pertinent target populations, the identification of which is a key step of such studies.

Autophagy is a catabolic process targeting cytosolic compounds to the lytic compartment after sequestration within a double membrane bound vesicle: the autophagosome. Along with the ubiquitin-proteasome pathway, autophagy is one of the main catabolic processes conserved among eukaryotic cells. Present at a basal level, it can be stimulated to allow: remobilization of cell resources, cytoprotective functions, and detoxification. Autophagosome formation demonstrates the capacity of the endomembrane system to adapt dynamically to the cell's environment. However, the membrane and molecular processes involved are still poorly understood. This work aimed to advance understanding of autophagosome formation in plant cells. First of all, we set up suitable conditions for the study of autophagy in the Arabidopsis root, then we identified markers of the autophagosome formation steps. Live and 3D imaging of the ATG5 protein, involved in membrane expansion, demonstrated its transient recruitment to a specific domain of the forming autophagosome, its aperture. Furthermore, studying different actors of the endomembrane system has allowed us to implicate the endoplasmic reticulum and ATG9, and to establish a model for autophagosome formation in plants.; L'autophagie est un processus permettant la dégradation de constituants cytosoliques dans un compartiment lytique, par leur séquestration au sein d'une vésicule à double membrane : l'autophagosome. L'autophagie est, avec la voie ubiquitine-protéasome, l'une des deux grandes voies de dégradation présente de manière fortement conservée chez les cellules eucaryotes. Présente à un niveau basal, elle peut être stimulée afin de permettre la remobilisation de ressources cellulaires, ou d'assurer des fonctions cytoprotectrices et de détoxification. La formation d'autophagosomes traduit alors la capacité du système endomembranaire à s'adapter aux besoins cellulaires. Cependant, la mécanique membranaire et moléculaire de ce phénomène reste mal comprise. L'objectif de ce travail de thèse était de mieux comprendre la formation de ce compartiment dans la cellule végétale. Pour cela, nous avons tout d'abord mis au point les conditions propices à l'étude de l'autophagie dans la racine d’Arabidopsis thaliana, puis nous avons entrepris l'identification de marqueurs des étapes de formation de l'autophagosome. L'étude par imagerie en temps réel et 3D de la protéine ATG5, impliquée dans l’expansion membranaire, nous a permis de mettre en évidence son recrutement transitoire sur un domaine particulier de l'autophagosome en formation, son ouverture. De plus, l'étude de différents acteurs du système endomembranaire, nous a permis de mettre en évidence et de caractériser l'implication du réticulum endoplasmique et de ATG9, pour aboutir à un modèle de la formation de l'autophagosome chez les plantes.

Autophagy is a catabolic process targeting cytosolic compounds to the lytic compartment after sequestration within a double membrane bound vesicle: the autophagosome. Along with the ubiquitin-proteasome pathway, autophagy is one of the main catabolic processes conserved among eukaryotic cells. Present at a basal level, it can be stimulated to allow: remobilization of cell resources, cytoprotective functions, and detoxification. Autophagosome formation demonstrates the capacity of the endomembrane system to adapt dynamically to the cell's environment. However, the membrane and molecular processes involved are still poorly understood. This work aimed to advance understanding of autophagosome formation in plant cells. First of all, we set up suitable conditions for the study of autophagy in the Arabidopsis root, then we identified markers of the autophagosome formation steps. Live and 3D imaging of the ATG5 protein, involved in membrane expansion, demonstrated its transient recruitment to a specific domain of the forming autophagosome, its aperture. Furthermore, studying different actors of the endomembrane system has allowed us to implicate the endoplasmic reticulum and ATG9, and to establish a model for autophagosome formation in plants.; L'autophagie est un processus permettant la dégradation de constituants cytosoliques dans un compartiment lytique, par leur séquestration au sein d'une vésicule à double membrane : l'autophagosome. L'autophagie est, avec la voie ubiquitine-protéasome, l'une des deux grandes voies de dégradation présente de manière fortement conservée chez les cellules eucaryotes. Présente à un niveau basal, elle peut être stimulée afin de permettre la remobilisation de ressources cellulaires, ou d'assurer des fonctions cytoprotectrices et de détoxification. La formation d'autophagosomes traduit alors la capacité du système endomembranaire à s'adapter aux besoins cellulaires. Cependant, la mécanique membranaire et moléculaire de ce phénomène reste mal comprise. L'objectif de ce travail de thèse était de mieux comprendre la formation de ce compartiment dans la cellule végétale. Pour cela, nous avons tout d'abord mis au point les conditions propices à l'étude de l'autophagie dans la racine d’Arabidopsis thaliana, puis nous avons entrepris l'identification de marqueurs des étapes de formation de l'autophagosome. L'étude par imagerie en temps réel et 3D de la protéine ATG5, impliquée dans l’expansion membranaire, nous a permis de mettre en évidence son recrutement transitoire sur un domaine particulier de l'autophagosome en formation, son ouverture. De plus, l'étude de différents acteurs du système endomembranaire, nous a permis de mettre en évidence et de caractériser l'implication du réticulum endoplasmique et de ATG9, pour aboutir à un modèle de la formation de l'autophagosome chez les plantes.

The primary objective in creating herbicide-tolerant varieties is to offer the farmers with an efficient tool to ensure weed control and solve weed problems, but their use could eventually have expected or unexpected consequences according to conditions of implementation. A few of those varieties, obtained either through classical breeding or through induced mutagenesis are now on the European market (especially oilseed rape and sunflower) while a majority, which was obtained through genetic engineering, is not allowed for cultivation in this area. The report describes how CNRS and INRA experts dealt with the scientific literature, extracts key elements, points out necessary watchfulness on potential impacts detected by the expertise, and considers their use in the framework of the French "Ecophyto 2018" plan that aims at reducing the use of agrochemicals., Les variétés tolérantes à un herbicide (VTH) visent d’abord à proposer aux agriculteurs une réponse technique à des difficultés de désherbage, mais leur usage peut avoir des effets attendus ou non selon leurs conditions d’emploi. Certaines de ces variétés issues de sélection classique ou de mutagénèse sont actuellement commercialisées en Europe (notamment colza et tournesol) alors que la majorité, obtenue par transgénèse, n’y est pas autorisée à la mise en culture. L’exposé rappelle la démarche de l’expertise réalisée par le CNRS et l’INRA fondée sur la bibliographie scientifique, extrait les éléments clés, souligne les points de vigilance soulevés par cette expertise et replace l’utilisation des VTH dans le cadre du plan Ecophyto 2018 dont l’objectif est de limiter les intrants phytosanitaires.

Opines are produced in plant cells transformed with the A. tumefaciens T-DNA. These opines can be used as nutrients by the phytopathogen and some of them act as signaling molecules controlling Ti plasmid dissemination through quorum-sensing. The present study aims at an enlarged understanding of the roles of opines during A. tumefaciens/plant host interactions. First, based on transcriptomic investigations, the agrocinopine-controled AccR regulon from A. tumefaciens C58 was thoroughly characterized. This one includes functions associated with (i) agrocinopines assimilation, (ii) nopaline assimilation, (iii) quorum-sensing and Ti plasmid conjugation, (iv) At plasmid conjugation. Moreover our analysis showed that co-regulation of Ti and At plasmid conjugations correlated with the co-transfer of the two replicons. In a second step using functional genetic and structural biology we quantified the selective advantage conferred to colonizing A. tumefaciens populations by the assimilation of the opines nopaline and octopine. The molecular basis which underlies this selective advantage, especially regarding the sensing and import of the two opines within the bacterial cytoplasm, were also described. Finally combining metabolomics and reverse genetic with in planta conjugation assays we demonstrated the opposite effects of GABA and opine/quorum-sensing signaling on the dissemination of Ti plasmids. In conclusion our results reveal the interlink between opine, quorum-sensing and GABA signaling during A. tumefaciens/plant host interactions. They noticeably highlight the impact of this interlink on host colonization and the dissemination of Ti and At plasmid genes which are critical for the virulence and the adaption of the bacteria to the tumor lifestyle., Les opines sont des molécules produites dans les cellules végétales transformées par l’ADN-T d’A. tumefaciens. Ces opines peuvent être utilisées comme nutriments par le phytopathogène et certaines d’entre elles agissent comme signaux moléculaires contrôlant la dissémination du plasmide de virulence Ti via la signalisation quorum-sensing. Le présent travail vise à une compréhension élargie du rôle des opines au cours des interactions A. tumefaciens-plantes hôtes. En se basant d’abord sur des analyses transcriptomiques, le régulon AccR d’A. tumefaciens C58, contrôlé par les opines agrocinopines, a été défini : celui-ci inclut des fonctions associées (i) à l’assimilation des agrocinopines, (ii) à l’assimilation de la nopaline, (iii) à la signalisation quorum-sensing et la conjugaison du plasmide Ti, (iv) à la conjugaison du plasmide At. La corrélation entre la co-régulation des conjugaisons des plasmides Ti et At et le co-transfert des deux réplicons a en outre été mise en évidence. Dans un second temps, associant des approches de génétique fonctionnelle à des travaux collaboratifs en biologie structurale, l’avantage sélectif conféré par les opines nopaline et octopine à A. tumefaciens au sein des tumeurs végétales a été quantifié. Les bases moléculaires sous-jacentes à cet avantage sélectif, notamment celles associées à la perception et l’importation des deux opines dans le cytoplasme bactérien, ont été décrites. Enfin, en combinant des approches de métabolomique et de génétique inverse avec des tests de conjugaison in planta, les effets opposés de la signalisation GABA d’une part et des signalisations opine et quorum-sensing d’autre part sur la dissémination du plasmide Ti ont été démontrés. En conclusion, nos résultats révèlent l’intrication des signalisations opine, quorum-sensing et GABA au cours de l’interaction A. tumefaciens-plantes hôtes. Ils soulignent en particulier les impacts de cette intrication sur la colonisation de l’hôte ainsi que sur la dissémination des gènes de virulence et d’adaptation à l’environnement tumeur portés par les plasmides Ti et At.

International audience; abstract simple

International audience; Bacteria belonging to the Pectobacterium genus are the causative agents of the blackleg and soft-rot diseases that affect potato plants and tubers worldwide. In Pectobacterium, the expression of the virulence genes is controlled by quorum-sensing (QS) and N-acylhomoserine lactones (AHLs). In this work, we screened a chemical library of QS-inhibitors (QSIs) and AHL-analogs to find novel QSIs targeting the virulence of Pectobacterium. Four N,N'-bisalkylated imidazolium salts were identified as QSIs; they were active at the µM range. In potato tuber assays, two of them were able to decrease the severity of the symptoms provoked by P. atrosepticum. This work extends the range of the QSIs acting on the Pectobacterium-induced soft-rot disease.

International audience; The semi-aquatic legumes belonging to the genus Aeschynomene constitute a premium system for investigating the origin and evolution of unusual symbiotic features such as stem nodulation and the presence of a Nod-independent infection process. This latter apparently arose in a single Aeschynomene lineage. But how this unique Nod-independent group then radiated is not yet known. We have investigated the role of polyploidy in Aeschynomene speciation via a case study of the pantropical A. indica and then extended the analysis to the other Nod-independent species. For this, we combined SSR genotyping, genome characterization through flow cytometry, chromosome counting, FISH and GISH experiments, molecular phylogenies using ITS and single nuclear gene sequences, and artificial hybridizations. These analyses demonstrate the existence of an A. indica polyploid species complex comprising A. evenia (C. Wright) (2n = 2x = 20), A. indica L. s. s. (2n = 4x = 40) and a new hexaploid form (2n = 6x = 60). This latter contains the two genomes present in the tetraploid (A. evenia and A. scabra) and another unidentified genome. Two other species, A. pratensis and A. virginica, are also shown to be of allopolyploid origin. This work reveals multiple hybridization/polyploidization events, thus highlighting a prominent role of allopolyploidy in the radiation of the Nod-independent Aeschynomene.

Symbiosis between Rhizobium bacteria and legumes leads to the formation of the root nodule. The endosymbiotic bacteria reside in polyploid host cells as membrane-surrounded vesicles where they reduce atmospheric nitrogen to support plant growth by supplying ammonia in exchange for carbon sources and energy. The morphology and physiology of endosymbionts, despite their common function, are highly divergent in different hosts. In galegoid plants, the endosymbionts are terminally differentiated, uncultivable polyploid cells, with remarkably elongated and even branched Y-shaped cells. Bacteroid differentiation is controlled by host peptides, many of which have antibacterial activity and require the bacterial function of BacA. Although the precise and combined action of several hundred host peptides and BacA has yet to be discovered, similarities, especially to certain insect-bacterium symbioses involving likewise host peptides for manipulation of endosymbionts, suggest convergent evolution. Rhizobium-legume symbiosis provides a rich source of information for understanding host-controlled endosymbiotic life in eukaryotic cells.

The plant's innate immune system detects potential biotic threats through recognition of microbe-associated molecular patterns (MAMPs) or danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR). A central regulator of pattern-triggered immunity (PTI) is the BRI1-associated kinase 1 (BAK1), which undergoes complex formation with PRR upon ligand binding. Although viral patterns inducing PTI are well known from animal systems, nothing similar has been reported for plants. Rather, antiviral defense in plants is thought to be mediated by post-transcriptional gene silencing of viral RNA or through effector-triggered immunity, i.e., recognition of virus-specific effectors by resistance proteins. Nevertheless, infection by compatible viruses can also lead to the induction of defense gene expression, indicating that plants may also recognize viruses through PTI. Here, we show that PTI, or at least the presence of the regulator BAK1, is important for antiviral defense of Arabidopsis plants. Arabidopsis bak1 mutants show increased susceptibility to three different RNA viruses during compatible interactions. Furthermore, crude viral extracts but not purified virions induce several PTI marker responses in a BAK1-dependent manner. Overall, we conclude that BAK1-dependent PTI contributes to antiviral resistance in plants.

Auxin is a key hormone concerning the control of plant physiology and the impact on plant development. Conditional plants for ABP1 allowed the post embryonic studies and have contributed to demonstrate the involvement of ABP1 in a broad range of cellular and developmental responses including the clathrin-dependent endocytosis and the regulation of Aux/IAAs homeostasis. These datas revealed that an ABP1-dependent pathway is acting on transcriptional regulation by modulating the SCFTIR/AFBs signaling pathway. I took advantage of the phenotype of dark grown seedlings to study cell expansion in ABP1 loss of function background. ABP1 knockdown induced modifications of fucosylated form of xyloglucan side chains that are the main hemicellulose in Arabidopsis primary cell wall. All data converge to show that this effect results from alterations of expression of cell wall related genes via the modulation of the SCFTIR/AFBs pathway. In parallel, I used a suppressor approach to discover new signaling components downstream of ABP1. Characterisation of one of the suppressor leads to the identification of a loss of function allele of DCL3. This data demonstrates the involvement of the RNA directed DNA methylation pathway downstream of ABP1., L’auxine est une hormone fondamentale dans le développement et la physiologie de la plante. L’obtention des plantes conditionnelles pour ABP1 a permis la mise en évidence de son importance dans la signalisation de l’auxine. Ainsi ABP1 agirait d’une part sur l’endocytose de vésicules à clathrine au niveau de la membrane plasmique et d’autre part sur la stabilité des Aux/IAAs. Ce dernier résultat suggère qu’une voie de signalisation en aval d’ABP1 permet de modifier l’homéostasie de la voie de régulation transcriptionnelle de l’auxine, la voie SCFTIR/AFBs.Mon travail de thèse a consisté à caractériser les plantes inactivées pour ABP1 lors de la croissance à l’obscurité dans la plante modèle Arabidopsis thaliana. Mon étude montre qu’ABP1 contrôle l’expansion cellulaire en jouant sur la plasticité pariétale. J’ai ainsi pu mettre en évidence une modification de la proportion de formes fucosylées des chaînes latérales des xyloglucanes, le principal hémicellulose de la paroi primaire chez Arabidopsis. Cette modification de la fucosylation des xyloglucanes requiert des changements d’expressions géniques médiés ce qui conforte l’existence d’une voie de signalisation reliant ABP1 à la voie SCFTIR/AFBs.En parallèle, j’ai mené une approche génétique de recherche de suppresseurs du phénotype lié à l’inactivation d’ABP1 à l’obscurité. Parmi les dix lignées validées, j’ai d’ores et déjà identifié le gène DCL3 comme étant impliqué dans la suppression du phénotype ss12k et mis en évidence l’implication de la voie d’extinction de gènes par l’intermédiaire de petits ARNs non codant (voie RdDM) dans le contrôle de l’expansion cellulaire.

International audience; Seeds are a crucial stage in plant life. They contain the nutrients necessary to initiate the development of a new organism. Seeds also represent an important source of nutrient for human beings. Iron (Fe) and zinc (Zn) deficiencies affect over a billion people worldwide. It is therefore important to understand how these essential metals are stored in seeds. In this work, Particle-Induced X-ray Emission with the use of a focused ion beam ($\mu$PIXE) has been used to map and quantify essential metals in Arabidopsis seeds. In agreement with Synchrotron radiation X-ray fluorescence (SXRF) imaging and Perls/DAB staining, $\mu$PIXE maps confirmed the specific pattern of Fe and Mn localization in the endodermal and subepidermal cell layers in dry seeds, respectively. Moreover, $\mu$PIXE allows absolute quantification revealing that the Fe concentration in the endodermal cell layer reaches $\sim$800 $\mu$g·g$^{−1}$ dry weight. Nevertheless, this cell layer accounts only for about half of Fe stores in dry seeds. Comparison between Arabidopsis wild type (WT) and mutant seeds impaired in Fe vacuolar storage ($vit1-1$) or release ($nramp3nramp4$) confirmed the strongly altered Fe localization pattern in $vit1-1$, whereas no alteration could be detected in $nramp3nramp4$ dry seeds. Imaging of imbibed seeds indicates a dynamic localization of metals as Fe and Zn concentrations increase in the subepidermal cell layer of cotyledons after imbibition. The complementarities between $\mu$PIXE and other approaches as well as the importance of being able to quantify the patterns for the interpretation of mutant phenotypes are discussed.

International audience; The dimension of organs depends on the number and the size of their component cells. Formation of polyploid cells by endoreduplication cycles is predominantly associated with increases in the cell size and implicated in organ growth. In plants, the CCS52A proteins play a major role in the switch from mitotic to endoreduplication cycles controlling thus the number of mitotic cells and the endoreduplication events in the differentiating cells. * Arabidopsis has two CCS52A isoforms; AtCCS52A1 and AtCCS52A2. Here we focused on their roles in endoreduplication and cell size control during plant development. We demonstrate their complementary and dose-dependent actions that are dependent on their expression patterns. Moreover, the impact of CCS52A overexpression on organ size in transgenic plants was dependent on the expression level; while enhanced expression of the CCS52A genes positively correlated with the ploidy levels, organ sizes were negatively affected by strong overexpression whereas milder overexpression resulted in a significant increase in the organ sizes. * Taken together, these finding support both complementary and dose-dependent actions for the Arabidopsis CCS52A isoforms in plant development and demonstrate that elevated ectopic CCS52A expression positively correlates with organ size, opening a route to higher biomass production.